Conditional sex transformation systems could improve genetic control strategies against insect pests. Here, we developed and tested CRISPR-based, tetracycline-repressible sex transformation strains in the Australian sheep blowfly, Lucilia cuprina. Using Tet-Off-regulated expression of Cas9 and dCas9, we targeted the sex-determining gene transformer with the goal of converting females into males. Conditional Cas9 expression enabled knockout of a visual marker gene, confirming inducible genome editing. However, strains expressing transformer-targeting sgRNA arrays did not undergo sex transformation. Embryonic microinjection of transformer-targeting sgRNAs into Cas9-expressing embryos produced intersex individuals, indicating that sgRNA expression from the integrated arrays was insufficient to disrupt the sex determination pathway. In contrast, high-level dCas9 expression was associated with developmental delays, reduced body weight, and lethality. These findings establish the first conditional CRISPR expression system in L. cuprina and demonstrate that Cas9 is compatible with inducible gene editing, whereas dCas9 is poorly tolerated at high expression levels.

The New World screwworm, Cochliomyia hominivorax, is an obligate parasite of warm-blooded animals and a major pest of livestock and wildlife in the Americas. The first genome assembly for C. hominivorax enabled substantial progress in key areas including gene expression related to fly behavior and physiology and gene editing technologies. However, the first genome was sequenced prior to several technological advances that result in fewer errors and better genome annotations. Here, we used the trio-binning approach to produce haplotype-resolved genome assemblies of C. hominivorax. A single male progeny from the cross of a Panama line male with a production strain female was sequenced using PacBio HiFi and scaffolded using Hi-C chromatin conformation, while Illumina NextSeq 2000 was used for short-read sequencing of both parents to facilitate trio-binning. We produced a linear haploid reference assembly by transferring a copy of the X chromosome and mitochondrial genome to the paternal haplotype. This assembly is comprised of 5 autosomes, 2 sex chromosomes, the mitogenome, and 75 unplaced scaffolds spanning 455.6 Mb, which is closer to the predicted size based on flow cytometry (443.8 Mb) than the previous assembly of 534.4 Mb. NCBI's external Eukaryotic Genome Annotation Pipeline (EGAPx) was used to annotate the protein-coding and non-coding genes in the linear haploid reference and the maternal haplotype assemblies. Due to the better resolution of the sex chromosomes and updated genome annotations, these improved assemblies will advance future experiments aimed at understanding sex determination, gene expression, and the evolution of parasitism in the Calliphoridae.

The New World screwworm, Cochliomyia hominivorax (Coquerel, 1858; Diptera: Calliphoridae), was eradicated from North and Central America through the first application of the sterile insect technique. The sterile screwworm adult fly releases were mixed sex, but releasing only males could be much more effective. Here, we describe screwworm transgenic sexing strains (TSSs) with expected embryo lethality. Strains were developed with a Tetracycline-off (Tet-off) system utilizing either the Lucilia cuprina (Wiedemann, 1830) nullo (DR6) or Cochliomyia macellaria (Fabricius, 1755) CG14427 (DR7) gene promoter and a tTA-activated effector to promote female lethality. The promoter activity was highest in 2 to 3 h embryos and low in larvae and adults. However, these strains also had unexpected high expression in pupae. Evaluation on two doxycycline (Dox) suppression regimens found that inclusion of Dox in the last larval feeding rescued females of the subsequent generation, likely by maternal transfer of Dox. All TSSs produced only males on a reduced Dox feeding regimen, but the female lethal period for the DR6 TSS was too late in development to reduce diet costs. Production parameters were met by all strains in colony, but strains had lower male fly survival than current production strains after removing Dox. In noncompetitive mating success trials, DR6 strains performed poorly, but DR7 performed equally to production strain males. However, males from all TSSs fared poorly in mating competition tests against production males. Our study highlights the importance of tightly regulated gene promoters and stage-specific antibiotic feeding schemes for the development and evaluation of TSSs based on the Tet-off system.

Abstract Drosophila suzukii (Matsumura, 1931, Diptera: Drosophilidae) is a globally invasive pest of soft-skinned fruits that is currently controlled largely through the use of broad-spectrum insecticides. Increasing resistance to insecticides and regulatory pressures have motivated the development of genetic control strategies. We previously developed a CRISPR/Cas9-based homing gene drive targeting the coding sequence of the female-specific exon of the sex-determination gene doublesex , achieving highly efficient inheritance (94–99%) in both male and female germlines. A major limitation of homing gene drives is the formation of resistant alleles that evade cleavage yet retain gene function. Multiplexing guide RNAs (gRNAs) could reduce the formation of such functional resistance alleles. Here, we generated and tested homing constructs expressing one, two, or three gRNAs targeting different regions of the female-specific exon of doublesex , including the intron-exon splice junction. A single gRNA targeting the splice junction supported high inheritance in males but showed reduced efficiency in females. Combining this gRNA with a coding sequence-targeting guide further reduced drive efficiency, particularly in the female germline. Constructs expressing two gRNAs performed similarly whether guides were linked by transfer RNA (tRNA) sequences or expressed from independent promoters. Constructs expressing three gRNAs using tRNA processing showed consistently low drive inheritance in both sexes and low frequencies of target-site modification among non-drive progeny, consistent with reduced cleavage activity. Inheritance was significantly higher in male than female germlines for several constructs, indicating that germline context strongly influences drive performance. Our findings show that the strategy used for multi-gRNA expression, target site choice and sex-specific germline environments can influence gene drive efficiency, emphasizing the need to optimize construct design in the target species. Author Summary Spotted wing drosophila ( Drosophila suzukii ) is an invasive pest that damages soft skinned fruits such as berries and cherries. Control currently relies heavily on insecticides, but resistance and regulatory concerns are increasing the need for alternative approaches. We are developing genetic strategies for suppression of pest populations. We previously developed a gene editing system targeting a female-essential gene that showed very high biased inheritance (gene drive). However, a key challenge is that the system can sometimes create resistant individuals that are unaffected. One possible solution is to use using multiple gRNAs—molecules that direct gene editing to specific DNA sequences—to reduce the formation of these resistant individuals. In this study we found that using more than one gRNA reduced the efficiency of inheritance, especially in females. However, activity could be influenced by exactly where the DNA is targeted and how the guide RNAs are expressed. These results show that gene drive performance depends strongly on biological context and design choices, and that strategies must be carefully optimized in the target species.

The blowfly Lucilia cuprina is a destructive parasite of sheep that causes flystrike or myiasis. Larvae consume the animal’s living flesh, producing large wounds that can lead to death. The main aim of this study was to identify genes that may play important roles in the behavior and physiology of L. cuprina larvae. An RNA-Seq analysis of RNA from whole larvae at different developmental stages and third-instar head and gut tissues was used to identify sensory receptors and other genes relevant to the physiology of L. cuprina larvae. In addition, CRISPR/Cas9 gene editing was used to obtain a loss-of-function mutation for the L. cuprina odorant coreceptor gene (LcupOrco). The response of mutant larvae and adult females to fresh and rotten meat at different temperatures was evaluated. The RNA-Seq analysis suggested that odorant (OR), gustatory, ionotropic, and Pickpocket receptors may not play a central role in the L. cuprina larval sensory signaling and digestive systems. Rather, ATP-binding cassettes (ABCs) were highly enriched in head and gut RNA, and odorant-binding proteins (OBPs) only in the head. To confirm that ORs are not essential for larval detection of rotten beef, diet-choice assays were performed including larvae and adults homozygous for a null mutation in LcupOrco. While the attraction of adult females to rotten beef was disrupted, LcupOrco mutant larvae showed no change in diet preference. The expression pattern of the ABC and OBP gene families suggests a central role in the sensory system of the L. cuprina larva for these receptors. Behavioral assays showed that ORs are essential for the adult female response to rotten beef, but not for larval behavior. These findings are consistent with high levels of expression of LcupOrco in the adult female antenna but very low expression in larvae.

This chapter reviews key few methods of genetic biocontrol focusing on those systems that have been developed for crop insect pests. These include “sterile release systems” where e.g. sterilized males produce sperm which carries dominant lethal mutations that result in their offspring failing to develop or prevents functional sperm from being produced. We contrast these systems with “fertile release systems” which involve releasing insects that are capable of producing some offspring but with genetic defects that e.g. mean that fewer females survive. The chapter discusses the varied strengths and weaknesses of these different systems.

The blowfly Lucilia cuprina dorsalis is a highly detrimental ectoparasite of sheep responsible for causing flystrike, a condition that can result in severe morbidity and potentially lead to death if left untreated. In contrast, Lucilia cuprina cuprina is necrophagous and not a pest. We have been interested in identifying the genes that may play a role in the evolution of a parasitic lifestyle in blowflies. The objective of the present work was to explore the physiological role of the L. cuprina long neuropeptide F gene (LcNPF) in L. c. cuprina. We used CRISPR/Ca9 to generate a strain carrying a loss-of-function knock-in mutation for LcNPF. An RNA-Seq analysis was performed, and physiological and behavioral assays were conducted to evaluate the role of LcNPF in larvae and adult flies. Our findings indicate that functional disruption of the LcNPF gene significantly impairs egg hatching, larval survival, weight gain, crawling speed and larval time to pupariation. These phenotypic changes were corroborated by RNA-Seq analysis, which revealed downregulation of transcripts in LcNPF null mutated larvae associated with the respective physiological processes. In contrast, the foraging behavior of the larvae under the tested conditions was not affected. Interestingly, NPF appears to be essential for the oviposition preference of females for rotten meat but not for male mating behavior or fertility. Our results suggest that NPF signaling plays a central regulatory role in multiple physiological processes across both the larval and adult stages. ### Competing Interest Statement The authors have declared no competing interest. U.S. National Science Foundation, https://ror.org/021nxhr62, DEB-2030345 Coordenação de Aperfeicoamento de Pessoal de Nível Superior, 001, 88887.373783/2019-00 Fundação de Amparo à Pesquisa do Estado de São Paulo, 2021/01641-6, 2022/08573-9

Lucilia cuprina, a species of blowfly, consists of two recognized subspecies: L. cuprina cuprina and L. cuprina dorsalis. Although they are morphologically and molecularly similar to each other, they have very different ecological roles. In Australia, L. c. dorsalis is predominantly found in rural areas and is the primary causative agent of sheep myiasis (flystrike), while L. c. cuprina is necrophagous and not a significant pest of livestock in the Americas or elsewhere. Here, we present a chromosome-scale genome assembly for L. c. cuprina and an improved assembly for L. c. dorsalis, enabling comparative genomic analysis between these subspecies. While both genomes share a similar gene content, subspecies-specific genes were identified, which may contribute to their divergent ecological roles -necrophagy in L. c. cuprina and parasitism in L. c. dorsalis. Phylogenetic analyses across target genomic regions reaffirm the close relationship between L. c. cuprina and L. c. dorsalis and position L. sericata as their sister species. Gene mutations linked to diazinon resistance were exclusively observed in L. c. dorsalis, whereas malathion resistance was detected in both subspecies. Additionally, we identified genes with accelerated evolutionary rates in each subspecies, which may underlie their distinct feeding behaviours. We also conducted a detailed analysis of chemosensory genes, revealing that L. c. dorsalis possesses slightly larger repertoires of all four chemosensory gene families studied. In comparison to Drosophila melanogaster, both subspecies exhibit an expanded gustatory receptor clade. Our findings provide valuable insights into the genetic factors underpinning parasitism and insecticide resistance and provide a valuable genetic resource for future research endeavours, including the development of engineered strains aimed at genetic biocontrol strategies. This work enhances our understanding of the evolutionary adaptations for this important blowfly species.

The application of CRISPR/Cas9 technology in Drosophila suzukii has become a powerful tool for advancing genetic engineering and pest control strategies. We recently developed phiC31 integrase self-docking lines that express phiC31 in the germline. Here we use recombination mediated cassette exchange to evaluate six Cas9 gene constructs at sites on chromosome 2 and the Y chromosome. The constructs use the germline promoters from the D. suzukii nanos, bag of marbles or 132tubulin genes with either the native 3'UTR from the same gene or the viral p10 3'UTR. Quantitative RT-PCR of RNA from testes and functional assays using Dswhite gRNA lines revealed significant differences in Cas9 expression among promoters and chromosomal locations. At the chromosome 2 site, the highest activity was obtained with the Dsnos and Dsbam promoters coupled with their native 3'UTRs. Y-linked genes displayed lower overall Cas9 expression compared to second chromosome lines with the highest expression obtained using the Dsnos promoter. However, a unique Y-linked line (YNCN1) harbored dual Dsnos-Cas9-Dsnos plasmid integrations, resulting in significantly higher expression of Cas9. Molecular analyses confirmed the stable line had likely resulted from two separate recombination events between the attB site in a plasmid and one of the attP sites. Our findings highlight the impact of promoter choice and chromosomal context on transgene expression in D. suzukii and suggest that multi-copy insertions can overcome the transcriptional repression on the heterochromatic Y chromosome. These insights provide valuable tools for developing Y-linked editor strains or sterile insect systems to manage D. suzukii populations.

Genetic biocontrol is an increasingly important way to suppress insect pest populations and to mitigate their economic and health impact. One key advantage is that it is species-specific, as it relies on the mating of released males with wild females to either suppress or modify populations. The latter is through rendering females incompetent at disease transmission. Sex separation is critical to ensure the efficiency of these control programs, and it is essential in the case of vector control to avoid releasing females that can transmit pathogens. Modern genetic methods provide the opportunity to target or manipulate components of the sex determination systems to facilitate genetic biocontrol with new means to effectively accomplish sex-specific selection, lethality, or sterility. For example, sex-specific splicing elements in genes in the sex determination pathway are used to produce sex-specific markers. Sex-linked recessive lethal alleles are used to differentially eliminate the transgene-marked sex chromosome from males to produce nontransgenic males. Knocking out or knocking down sex-specific isoforms of genes in the sex determination pathway is employed to confer female-specific lethality or sterility. Sex determination pathways and sex chromosomes are also targeted for gene drives that suppress pest populations by introducing extreme sex ratio biases. Here, we review these and other recent advances in genetic technologies for pest control that have benefited from knowledge of sex determination systems in Diptera.

The New World screwworm is an obligate parasitic fly and a significant economic pest of livestock in the Americas. Although eradicated from the USA using the Sterile Insect Technique (SIT), enhancing SIT efficiency remains a priority. A promising approach involves conditional female-lethal genetic strains that produce only males in the absence of tetracycline, ideally eliminating females early in development to reduce larval diet costs. However, while some strains match wild-type production levels, lower male fitness reduces the net benefit of replacing the current wild-type strain with one of these genetic-sexing strains. This study aimed to improve strain performance through female-specific expression of both the driver and effector components of the lethality system. We tested four transgenic strains using early embryo-specific promoters from the Chhalo and g6451 genes. Strains with the Chhalo promoter driving tTA expression exhibited early-stage female lethality under a modified doxycycline regimen but suffered from reduced male fitness. In contrast, one strain with the g6451 promoter produced males with excellent fitness but female lethality occurred at the late pupal stage. Despite imperfect female lethality timing, the overall fitness characteristics of this strain makes it a good candidate for future sterile or fertile male release genetic control programs.

Many eye colour mutants have been identified in Drosophila melanogaster. Mutations in the sepia gene result in brown eyes due to a lack of PDA synthase, which is essential for production of the red drosopterin eye pigment. We previously used CRISPR/Cas9 to target the PDA synthase gene to establish sepia mutant strains for Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), an invasive global pest of soft skinned fruits. The fecundity and fertility of some of the sepia mutant strains were similar to wild-type. The goal of this study was to determine if the sepia gene could be used as a marker to identify transgenic D. suzukii. By using the sepia gene as a marker, we successfully developed lines expressing Streptomyces phage phiC31 integrase in the germline. For most of these lines, hemizygotes exhibited complete rescue of the sepia eye colour and relatively high levels of phiC31 RNA in ovaries. In contrast, lines with partial rescue showed low levels of sepia RNA in heads and phiC31 RNA in ovaries. These findings suggest that the sepia gene is an effective marker for D. suzukii transgenesis and its relatively small size (1.8 kb) makes it advantageous when assembling large gene constructs. The phiC31 integrase lines established in this study should serve as a valuable resource for future genetic research in D. suzukii, including the further development of strains for genetic biocontrol.

Blow flies (Diptera: Calliphoridae) occur worldwide and exhibit a wide range of larval feeding habits, including saprophagy, coprophagy, parasitism and predation. Understanding their biology is critical for medical and veterinary science and ecology. Calliphorids thrive across a range of habitats and exhibit complex life histories, with larvae developing immersed in their food substrate, while adults are free-living and have diverse feeding strategies. Some species have evolved specialized parasitic associations with vertebrate or invertebrate hosts, which are behaviors with important implications for agriculture and for understanding evolutionary transitions between saprophagy and parasitism. This study presents a comprehensive phylogenetic analysis of the Calliphoridae, utilizing 711 of 736 analysed nuclear genes, using anchored hybrid enrichment, from a global collection of blow flies and their relatives. Our results provide a robust and novel reconstruction of the evolutionary history of this group, pinpointing major transitions in larval feeding habits. We argue that saprophagy evolved independently multiple times from invertebrate parasitic ancestors, with vertebrate parasitism emerging from a number of different feeding strategies. These findings challenge prior hypotheses and offer new insights into the adaptive traits driving trophic specialization and diversification in this group.

Tetracyclines are broad-spectrum antibiotics widely used in agriculture, medicine, and research. However, they are associated with harmful side effects. In arthropods, parental exposure to tetracyclines has been linked to reduced health and fitness in untreated offspring. These transgenerational effects of tetracyclines could jeopardize the success of pest control programs that use tetracyclines to control gene expression. In this study, we investigated the transgenerational effects of 2 tetracyclines, doxycycline (DOX) and anhydrotetracycline (ATC), in the blowfly Lucilia cuprina, a significant pest of sheep. To simulate the rearing conditions of a transgenic male-only release program, blowflies were reared on standard diet alone, or standard diet plus DOX or ATC, for 3 generations, and then reared for an additional fourth generation on standard diet alone. We used behavioral assays, 16S amplicon sequencing, and mRNA sequencing to determine how DOX and ATC influenced male sexual competitiveness, microbiome composition, and gene expression in the third and fourth generations. We found that 3 generations of DOX treatment led to lower sexual competitiveness in both third- and fourth-generation males. In addition, DOX and ATC shifted the composition of the blowfly microbiome and altered the expression of numerous mitochondria- and immunity-related genes in both generations. Our study supports an emerging body of evidence that tetracyclines exert not only direct but also transgenerational effects, and sheds light on the transcriptional and microbial responses to antibiotic exposure and removal. Our findings emphasize the need for pest control programs that use tetracyclines to evaluate the long-term effects of these antibiotics.

Conditional female-to-male sex conversion systems are promising tools for improving the Sterile Insect Technique, an environmentally-friendly form of genetic pest control. In recent years, several conditional sex conversion systems, employing various effector genes and gene expression techniques, have been designed and evaluated in diverse insect species. While no system described thus far is ready for real-world use, valuable insight into insect physiology and sex determination has been gained. Additional basic research on insect sex determination mechanisms, particularly dosage compensation, coupled with increasingly flexible and powerful tools for gene expression and editing, should enable researchers to improve existing sex conversion systems, as well as to develop new systems in non-model insect pests.

Background Hyperglycemia influences the development of glomerular endothelial cell damage, and nowhere is this more evident than in the progression of diabetic kidney disease (DKD). While the Set7 lysine methyltransferase is a known hyperglycemic sensor, its role in endothelial cell function in the context of DKD remains poorly understood. Methods Single-cell transcriptomics was used to investigate Set7 regulation in a mouse model of DKD, followed by validation of findings using pharmacological and short hairpin RNA inhibition inhibition of Set7. Results Set7 knockout (Set7KO) improved glomerular structure and albuminuria in a mouse model of diabetes. Analysis of single-cell RNA-sequencing data showed dynamic transcriptional changes in diabetic renal cells. Set7KO controls phenotype switching of glomerular endothelial cell populations by transcriptional regulation of the insulin growth factor binding protein 5 (IGFBP5). Chromatin immunoprecipitation assays confirmed that the expression of the IGFBP5 gene was associated with mono- and dimethylation of histone H3 lysine 4 (H3K4me1/2). This generalizability was investigated in human kidney and circulating hyperglycemic cells exposed to TGF beta 1. We showed that the highly selective Set7 inhibitor (R)-PFI-2 hydrochloride attenuated indices associated with renal cell damage and mesenchymal transition, specifically (1) reactive oxygen species production, (2) IGFBP5 gene regulation, and (3) expression of mesenchymal markers. Furthermore, renal benefit observed in Set7KO diabetic mice closely corresponded in human glomerular endothelial cells with (R)-PFI-2 hydrochloride inhibition or Set7 short hairpin RNA silencing. Conclusions Set7 regulates the phenotypic endothelial-mesenchymal transition switch and suggests that targeting the lysine methyltransferase could protect glomerular cell injury in DKD

Tissue-specific gene promoters are desired as they provide the specificity needed for control of gene expression in transgenic animals. Here we describe a relatively rapid two-component transient expression assay that was used to identify a gene promoter active in the larval salivary glands of the green blow fly, Lucilia sericata. Sterile L. sericata maggots are widely used for wound debridement. A larval salivary gland gene promoter could be used to make maggots that secrete factors for enhanced wound therapy. Embryos from a line that carry a tetracycline transactivator (tTA)-activated red fluorescent protein gene were injected with plasmid DNA with the tTA gene driven by a constitutive or tissue-specific gene promoter. The hatched larvae were reared on diet and then examined for red fluorescence. A promoter from the LsCG30371 gene was active in the larval salivary glands. The tissue-specificity of the promoter was subsequently confirmed with stable transgenic lines that carried the LsCG30371-tTA gene. The relatively rapid transient expression assay could potentially be used to determine the tissue-specificity of other gene promoters. Further, the stable LsCG30371-tTA lines could be used to make sterile maggots that secrete factors from the salivary glands for enhanced wound healing.

Abstract Despite advances in the treatment of atherosclerotic cardiovascular disease, it remains the leading cause of death in patients with diabetes. Even when risk factors are mitigated, the disease progresses, and thus newer targets need to be identified that directly inhibit the underlying pathobiology of atherosclerosis in diabetes. A single cell sequencing approach was utilised to distinguish the proatherogenic transcriptional profile in aortic cells in diabetes using a streptozotocin induced-diabetic Apoe-/- mouse model. Human carotid endarterectomy specimens from individuals with and without diabetes were also evaluated via immunohistochemical analysis. Further mechanistic studies were performed in human aortic endothelial cells and human THP-1 derived macrophages. We then performed a preclinical study using an AP-1 inhibitor in a diabetic Apoe-/- mouse model. Single cell RNA sequencing analysis identified the AP-1 complex as a novel target in diabetes-associated atherosclerosis. AP-1 levels were elevated in carotid endarterectomy specimens from diabetic when compared to non-diabetic individuals. AP-1 was validated as a mechanosensitive transcription factor via immunofluorescence staining for regional heterogeneity of endothelial cells of the aortic region exposed to turbulent blood flow and by performing microfluidics experiments in HAECs. AP-1 inhibition with T-5224 blunted endothelial cell activation as assessed by a monocyte adhesion assay and expression of genes relevant to endothelial function. Furthermore, AP-1 inhibition attenuated foam cell formation. Critically, treatment with T-5224 attenuated atherosclerosis development in diabetic Apoe-/- mice. This study has identified the AP-1 complex as a novel target, inhibition of which treats the underlying pathobiology of atherosclerosis in diabetes. Article Highlights · Cell-specific transcriptional profile of vascular cells in diabetes-associated atherosclerosis is not known. · Single cell sequencing analysis not only defined the transcriptional profile of vascular cells in diabetes but also identified the AP-1 complex as one of the important transcription factor complexes in flow mediated endothelial cell activation and foam cell formation. · Although, previous studies have implicated AP-1 in atherosclerosis, our study has defined the role of AP-1 as a central regulator of a gene expression program linked to endothelial dysfunction and foam cell formation in diabetes. · Importantly, inhibition of AP-1 transcription activity with T-5224 attenuated atherosclerosis development in vivo in diabetes.

β-cells are a type of endocrine cell found in pancreatic islets that synthesize, store and release insulin. In type 1 diabetes (T1D), T-cells of the immune system selectively destroy the insulin-producing β-cells. Destruction of these cells leads to a lifelong dependence on exogenous insulin administration for survival. Consequently, there is an urgent need to identify novel therapies that stimulate β-cell growth and induce β-cell function. We and others have shown that pancreatic ductal progenitor cells are a promising source for regenerating β-cells for T1D owing to their inherent differentiation capacity. Default transcriptional suppression is refractory to exocrine reaction and tightly controls the regenerative potential by the EZH2 methyltransferase. In the present study, we show that transient stimulation of exocrine cells, derived from juvenile and adult T1D donors to the FDA-approved EZH2 inhibitors GSK126 and Tazemetostat (Taz) influence a phenotypic shift towards a β-like cell identity. The transition from repressed to permissive chromatin states are dependent on bivalent H3K27me3 and H3K4me3 chromatin modification. Targeting EZH2 is fundamental to β-cell regenerative potential. Reprogrammed pancreatic ductal cells exhibit insulin production and secretion in response to a physiological glucose challenge ex vivo. These pre-clinical studies underscore the potential of small molecule inhibitors as novel modulators of ductal progenitor differentiation and a promising new approach for the restoration of β-like cell function.